Our research goals are directed toward elucidation of the enzymatic mechanisms involved in the formation and degradation of neuropeptides. In the past several years we have identified, isolated and characterized several such enzymes in brain and pituitary. Our studies on the specificity and mechanism of action of these enzymes have led us to synthesize potent active-site directed inhibitors. Neuropeptide metabolizing enzymes represent an attractive target for pharmacological intervention. Inhibitors of such enzymes may represent a new generation of psychotherapeutic agents. This application proposes to continue and extend our studies on: prolyl endopeptidase, an enzyme that cleaves bonds after proline residues in neuropeptides, pyroglutamyl peptide hydrolases that remove the N-terminal pyroglutamyl residue from such peptides as thyrotropin releasing hormone (TRH), and the multicatalytic protease complex potentially involved in the metabolism of neuropeptides. Initial studies will concentrate on TRH. Similar studies will be conducted on other neuropeptides. Degradation of TRH proceeds through two pathways. Deamidation is catalyzed by prolyl endopeptidase and hydrolysis of the pyroglutamylhistidyl bond is catalyzed in part by pyroglutamyl peptide hydrolase. We have synthesized potent and specific active-site directed inhibitors of these two enzymes. Studies with these inhibitors have led to the discovery of a third TRH-degrading membrane-bound enzyme, highly localized to brain. This enzyme which may control the degradation of neuronally released TRH also cleaves the pyroglutamyl-His bond. It will be purified to homogeneity and characterized. An active-site directed inhibitor of this enzyme will be synthesized. Inhibitors of the three TRH degrading enzymes will be used to determine their role in the control of TRH levels and function in brain and pituitary in vivo. The concept that enzymatic degradation of neuropeptides may control their function will be tested in a pituitary GH-3 cell line culture which secretes prolactin in response to TRH. Studies will be continued on the multicatalytic protease complex discovered in our laboratory. Special attention will be directed toward studies on the component of the complex that is dramatically activated by low concentrations of sodium dodecyl sulfate and several natural fatty acids. Anti-sera obtained in our laboratory against prolyl endopeptidase and the multicatalytic protease complex will be used in a collaborative study to localize these enzymes in the CNS.